Proteins are polymers of amino acids which have biological activity. Often a protein will possess several distinct activities. It would be advantageous to have a systematic approach for determining where the functional parts are located. These parts could then be synthesized from recombinant DNA plasmids and used as portable pieces which have a specific biological activity. A major advantage to the use of functional pieces is that they are often much smaller than the full sized protein from which they are extracted. This reduction in size is of great advantage in certain applications. For example, with small pieces it would be possible to join several together to produce a polyprotein with a set of functional activities in a combination that does not occur naturally. This kind of construction would probably be limited to sequences of 1,000 or fewer amino acids since this is the upper limit of naturally occurring polypeptide chains. Utilizing small pieces, the sizes of newly constructed polyproteins may be limited to those which may be handled by the appropriate cellular machinery. For example, DNA sequences encoding only the short functional pieces could be inserted into plasmids which are limited as to the number of nucleotides which may be incorporated.
Most previous work on mapping protein domains has relied on protease digestion of the native protein. These digestions can reduce the size of proteins, but it is almost impossible to control the point at which the digestion stops. Usually, a limited, stable fragment is obtained and the activity of this limited piece is tested. It is not possible to make internal deletions in a protein using a protease since the proteases act by digesting amino acid residues, and do not rejoin them to produce new polypeptide sequence arrangements.
In order for polypeptide sequences to be assembled to form the new protein, it must first be shown that the functional regions may be separated from the remainder of the protein and retain their functional activity. Secondly, a method for deleting specific portions of the protein must also be available. In addition, a means for reassembling the polypeptide sequences and expression of the protein must be available.
In considering the problem of assembly of the protein, it would be advantageous to be able to isolate and synthesize pieces with oligomerization activity for use in forming new quaternary structures. This may even further alter the functional activity of the protein.
A still further aspect of the formation of new protein molecules involves assembly of pieces which are themselves inactive but which have catalytic or other functional activity when joined together. In the general context of enzyme structure-function, two broad types of complementation or interactions have previously been observed. One, two variants of the same enzyme, each with a different modification or mutatation in the active site region, can interact and thereby restore activity. The classical studies of active site histidines in ribonuclease are an example, described by A. M. Crestfield, W. H. Stein, and S. Moore in J. Biol. Chem., 238, 2413-2420 (1973 a) and J. Bol. Chem., 238, 2421-2428 (1963). Two, mutant and wild-type polypeptides can associate to give inactive oligomers. There are classes of mutations that are dominant defective, as a result of formation of mixed-subunit tetramers between defective and wild-type proteins. Examples in the lacl and recA systems are described by Davis and Jacob in J. Mol. Biol. 36, 413-417 (1968), Mueller-Hill et al, Proc. Nat. Acad. Sci. U.S.A. 59, 1259-1264 (1968), Geisler and Weber, Proc. Nat. Acad. Sci. U.S.A. 73, 3103-3106 (1976), and Yarranton and Sedgwick in Mol. Gen. Genet. 185, 99-104 (1982).
It would be advantageous to have a method, such as one using readily synthesized, inactive polypeptide fragments, for reactivating the activity-deficient protein through a specific protein-protein contact that is well removed from the functional region. Such a method is not presently available.
It is therefore an object of the invention to provide a method for mapping multiple functional domains in a protein or polypeptide sequence.
Another object of the invention is to provide a method for creating hybrid polypeptides with one or more specific functional activities.
A still further object of the invention is to provide a method for making a nested set of overlapping deletions within the carboxyl terminal encoding portions of a gene for a specific protein so that the deleted gene may be inserted into recombinant DNA plasmids for transformation into an appropriate host strain and expression of the desired protein.
Another object of the invention is to provide a method for identifying, isolating and expressing nucleotide sequences which encode polypeptides with adhesive or coupling properties.
Yet another object of the invention is to map and isolate gene and amino acid sequences for the functional components of alanine-tRNA synthetase, specifically those for adenalate synthesis, transfer RNA interactions, and oligomerization.